金黄色葡萄球菌杀白细胞素基因敲除菌株的构建及生长特性分析  

Construction and growth characteristics analysis of Panton-Valentine Leukocidin deletion mutant of Staphylococcus aureus

作　　者：刘文宇 祝瑶[1] 王长珍 杨亲 栾天 王凌利 刘思国[1] 周学章[2] 张万江[1] LIU Wen-yu;ZHU Yao;WANG Chang-zhen;YANG Qin;LUAN Tian;WANG Ling-li;LIU Si-guo;ZHOU Xue-zhang;ZHANG Wan-jiang(Division of Bacterial Diseases,State Key Laboratory of Veterinary Biotechnology,Harbin Veterinary Research Institute,Chinese Academy of Agriculture Science,Harbin 150069,China;School of life Science,Ningxia University,Yinchuan 750021,China)

机构地区：[1]中国农业科学院哈尔滨兽医研究所兽医生物技术国家重点实验室/动物细菌病创新团队,黑龙江哈尔滨150069 [2]宁夏大学生命科学学院,宁夏银川750021

出　　处：《中国预防兽医学报》2020年第11期1104-1108,共5页Chinese Journal of Preventive Veterinary Medicine

基　　金：国家重点研发计划(2017YFD0500102、2016YFD0501304);黑龙江省自然科学基金项目(YQ2019C031)。

摘　　要：为构建金黄色葡萄球菌杀白细胞素(PVL)基因敲除的菌株,本研究以pBT2温敏型质粒为载体,构建金黄色葡萄球菌PVL基因敲除质粒pBT2-ΔPVL,并经酶切鉴定。将其转化至金黄色葡萄球菌RN4220中经其修饰后电转化至金黄色葡萄球菌ATCC 49775中,在30℃和42℃多次交替传代并经红霉素抗性筛选,构建PVL基因敲除菌株,经PCR和测序鉴定后命名为ΔPVL。以pLI50穿梭质粒为载体,构建金黄色葡萄球菌PVL基因回补质粒pLI50-PVL,按照同样方法将其转化至敲除菌株ΔPVL中,构建PVL基因回补菌株CΔPVL。将ΔPVL在TSB培养基中连续传代培养后,经PCR检测敲除菌株体外的遗传稳定性;分别绘制敲除菌株ΔPVL和回补菌株CΔPVL的生长曲线,检测其体外生长特性。结果显示,正确构建了两种质粒pBT2-ΔPVL、pLI50-PVL和两种菌株ΔPVL、CΔPVL。敲除菌株ΔPVL连续传代未发生回复突变,具有良好的遗传稳定性。生长特性结果显示敲除菌株生长速率显著低于野生菌株,表明PVL基因在金黄色葡萄球菌的生长调节中发挥着一定的作用。本研究为进一步研究金黄色葡萄球菌PVL的功能及致病机制奠定了基础。In order to construct the panton-valentine leukocidin(PVL)deletion mutant of Staphylococcus aureus,temperaturesensitive plasmid PBT2 was used as vector to construct a S.aureus PVL knockout plasmid pBT2-ΔPVL.pBT2-ΔPVL modified by S.aureus RN4220 was electroporated into S.aureus ATCC 49775 and passaged multiple times at 30℃and 42℃alternately.The deletion mutant was identified by PCR and sequencing,and its genetic stability and growth characteristics were evaluated.The results showed that the PVL deletion mutant of S.aureus ATCC 49775 was successfully constructed and named asΔPVL.Using the pLI50 plasmid as a vector,construct the anapling plasmid pLI50-PVL for complementing the PVL gene of S.aureus.After restriction enzyme identification,transform it intoΔPVL according to the same method.After being identified by PCR and sequencing,the obtained replenishing strain was named CΔPVL.The result of genetic stability tests in vitro showed that the deletion mutant did not undergo reversion mutation in successive passages and had good genetic stability.The result of growth characteristics showed that the growth rate of the deletion mutant was significantly lower than that of the wild type strain,the growth rate of the replenishing strain is basically the same as that of the wild strain,indicating that the PVL gene plays a certain role in the growth regulation of S.aureus.This study laid the foundation for further study about the function and pathogenic mechanism of PVL in S.aureus.

关 键 词：杀白细胞素 同源重组 遗传稳定性 

分 类 号：S852.61[农业科学—基础兽医学]